Protocol for Low Input RNA: cDNA Synthesis and Amplification (NEB #E6421)

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	This is a point where you can safely stop the protocol and store the samples prior to proceeding to the next step in the protocol.
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Sample Recommendations

This protocol is to be used for total RNA. 

The RNA sample should be free of salts (e.g., Mg²+, or guanidinium salts), divalent cation chelating agents (e.g. EDTA, EGTA, citrate), or organics (e.g., phenol and ethanol). If an excess amount of genomic DNA is present in RNA samples, an optional DNase I treatment could be peformed. Inactivate/remove DNase I after treatment.

 Assess quality of the input RNA by running input RNA on an Agilent Bioanalyzer to determine the RNA Integrity Number (RIN).

Starting Material 
2 pg–200 ng poly(A) tail-containing total RNA (DNA free), RIN score > 8.0.

Typical Yield of cDNA from a Reaction 
Actual yields will depend on the quality and quantity of the input RNA, the mRNA content of the sample, and the method used to purify the RNA. Typical cDNA yields range between 5–15 ng (for the lower RNA inputs) based on the PCR cycle recommendations provided in Section 2.4.  

Notes
Keep all buffers and enzymes on ice, unless otherwise indicated.

2.1. Sample and Reagents Preparation

2.1.1. Briefly centrifuge the tubes containing NEBNext Single Cell RT Enzyme Mix and Murine RNase Inhibitor to collect solution to the bottom of the tube, then place on ice. 

2.1.2. Thaw all other frozen components at room temperature, (if the 10X NEBNext Cell Lysis Buffer appears cloudy after thawing, incubate briefly at 37°C to clear up the solution). 

2.1.3. Mix each component thoroughly, centrifuge briefly to collect solutions to bottom of the tubes, then place on ice. Leave the 10X NEBNext Cell Lysis Buffer at room temperature.

2.1.4. Thaw total RNA on ice prior to starting the protocol.

2.2 Primer Annealing for First Strand Synthesis

2.2.1. To anneal cDNA Primer with total RNA samples, prepare the reaction as follows (on ice): 

Component	< 5 ng RNA VOLUME (μl) PER RXN	≥ 5 ng RNA VOLUME (μl) PER RXN
Total RNA	Up to 8 µl	Up to 7 µl
(lilac) NEBNext Single Cell RT Primer Mix	1 µl	2 µl
Nuclease-free Water	Variable	Variable
Total Volume	9 µl	9 µl

2.2.2. Mix gently by pipetting up and down at least 10 times, then centrifuge briefly to collect solutions to the bottom of tubes.

2.2.3. Incubate for 5 minutes at 70°C in a thermocycler with the heated lid set to 105°C, then hold at 4°C until next step.

During the above annealing step, prepare the components for the following step.

2.3. Reverse Transcription (RT) and Template Switching

2.3.1. Vortex the NEBNext Single Cell RT Buffer briefly then prepare the RT mix in a separate tube as follows (adding NEBNext Single Cell RT Enzyme Mix last). 

Note: It is important to vortex the buffer prior to use for optimal performance.

COMPONENT	VOLUME (μl) PER REACTION
(lilac) NEBNext Single Cell RT Buffer	5 µl
(lilac) NEBNext Template Switching Oligo	1 µl
(lilac) NEBNext Single Cell RT Enzyme Mix	2 µl
Nuclease-free Water	3 µl
Total Volume	11 µl

2.3.2. Mix thoroughly by pipetting up and down several times, then centrifuge briefly to collect solutions to the bottom of tubes. 

2.3.3. Combine 11 µl of the RT mix (above) with 9 µl of the annealed sample (Step 2.2.3), mix well by pipetting up and down at least 10 times, and centrifuge briefly. 

2.3.4. Incubate the reaction mix in a thermocycler with the following steps and the heated lid set to 105°C:

90 minutes at 42°C
10 minutes at 70°C
Hold at 4°C

 Safe Stopping Point: Samples can be safely stored overnight at 4°C or –20°C.

2.4 cDNA Amplification by PCR

2.4.1. Prepare cDNA amplification mix as follows:

COMPONENT	VOLUME(μl) PER REACTION
(orange) NEBNext Single Cell cDNA PCR Master Mix	50 µl
(orange) NEBNext Single Cell cDNA PCR Primer	2 µl
(white) NEBNextCell Lysis Buffer (10x)	0.5 µl
Nuclease-free Water	27.5 µl
Total Volume	80 µl

2.4.2. Add 80 µl cDNA amplification mix to 20 µl of the sample from Step 2.3.4, mix thoroughly by pipetting up and down at least 10 times.

2.4.3. Incubate the reaction in a thermocycler with the following PCR cycling conditions and the heated lid set to 105°C:

Recommended Number of PCR Cycles

*Note: The amount of RNA in your sample should be used to determine the approximate number of PCR cycles.

For the various input listed above the recommended amplification PCR cycles will typically result in cDNA yields between 5–15 ng. The higher RNA input (> 100 ng) may yield > 15 ng cDNA. The total RNA used for the above recommendations is Universal Human Reference (UHR) RNA.  

 Safe Stopping Point: Samples can be safely stored overnight at 4°C or –20°C.

2.5. Cleanup of Amplified cDNA

2.5.1. Allow the NEBNext Bead Reconstitution Buffer and the SPRI beads (if stored at 4°C) to warm to room temperature for at least 30 minutes before use. Vortex SPRI beads to resuspend well and prepare fresh 80% ethanol.

2.5.2. Add 60 µl (0.6X of sample volume) resuspended beads to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Alternatively, samples can be mixed by vortexing for 3–5 seconds on high. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.

2.5.3. Incubate samples on the bench top for at least 5 minutes at room temperature.

2.5.4. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand. 

2.5.5. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA (Caution: do not discard the beads). 

2.5.6. Add 200 µl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.

2.5.7. Repeat Step 2.5.6 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol. 

2.5.8. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open. 

Caution: Do not over-dry the beads. This may result in lower recovery of cDNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

2.5.9. Remove the tube/plate from the magnetic stand. Elute the cDNA from the beads by adding 50 µl of 0.1X TE (dilute 1X TE Buffer 1:10 in water).

2.5.10. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube.

2.5.11. Add 45 µl of (room temperature) NEBNext Bead Reconstitution Buffer to the eluted cDNA + bead mixture from Step 2.5.10 for a second sample clean up. Mix well by pipetting up and down at least 10 times (Caution: Skipping this additional cleanup step may reduce overall cDNA purity).

2.5.12. Incubate samples on the bench top for at least 5 minutes at room temperature.

2.5.13. Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand. 

2.5.14. After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA (Caution: do not discard the beads). 

2.5.15. Add 200 µl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain cDNA.

2.5.16. Repeat Step 2.5.15 once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol. 

2.5.17. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open. 

Caution: Do not over-dry the beads. This may result in lower recovery of cDNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry.

2.5.18. Remove the tube/plate from the magnetic stand. Elute the cDNA from the beads by adding 33 μl of 1X TE (provided in kit). Incubate at room temperature for 2 minutes to rehydrate the beads. (Note: if the downstream library construction protocol recommends that DNA be in solution free of EDTA, elute the cDNA in 10 mM Tris (pH 8.0) instead of TE). 

2.5.19. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 minutes at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand. 

2.5.20. Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 30 µl to a new PCR tube. 

 Safe Stopping Point: Samples can be safely stored overnight at 4°C or –20°C.

Note: For subsequent library preparation protocols, we recommend that the DNA be in 1X TE (10 mM Tris pH 8.0, 1 mM EDTA), however, 10 mM Tris pH 7.5–8, low EDTA TE or H₂O are also acceptable.

2.6. Assess Amplified cDNA Quality and Quantity on a Bioanalyzer

2.6.1. Run 1 µl of amplified cDNA from Step 2.5.20 on a DNA High Sensitivity Chip.

Figure 2.6.1: Example of cDNA size distribution on a Bioanalyzer.